A major goal in neuroscience is to understand the formation and development of synapses, the tiny membrane specializations that enable nerve cells to communicate with each other. The sequence of molecular signals leading to synapse formation (synaptogenesis) is qualitatively well known for the more accessible neuromuscular junction (NMJ) [2]. However, very little is known of the quantities (concentration, duration, onset, etc.) of the various neurochemical signals involved in synaptogenesis. Intriguingly, all but one of the axons innervating a given myotube at birth retract after a period of ~1 week as a result of a synaptic competition process that remains, for lack of quantitative methods, poorly understood. Our overall objective is to uncover some of the rules governing the formation and elimination of synapses at the NMJ using a microfluidic cell culture system developed under a previous R01 (which we seek to renew for the first time). Our approach is based on substituting the presynaptic neuron by an artificial microfluidic device that delivers known doses of various synaptogenic neurochemicals to micrometer-scale areas of the membrane of cultured myotubes. We will focus on the three key factors - agrin, neuregulin, and the neurotransmitter acetylcholine (ACh) - secreted by the nerve tip during synaptogenesis. We will measure muscle cell responses that are specific to ACh receptors (AChRs), such as AChR aggregation/disaggregation, degradation/synthesis, insertion, co-localization with other receptors and cytoskeletal proteins, intracellular signaling pathways, etc. Under previous support, we have developed a microfluidic mimic of the innervation process that allows for focally stimulating >80 single, isolated (microengineered) myotubes using laminar flow streams (orthogonal to the myotubes). We have found that a) focal application of agrin entices myotubes to recruit new AChRs to the stimulated area; b) when the microtracks are formed with Matrigel, a basal lamina extract, the microengineered myotubes display AChR clusters of complex, in-vivo-like morphologies even before agrin is applied, similarly to what happens in vivo; and c) when agrin is focally applied to those agrin-predating clusters, AChRs are degraded at reduced rates, suggesting that a putative role for agrin in vivo is to help stabilize AChRs. We seek to continue these investigations by studying the dynamics and spatial patterns of various AChR-specific responses upon (competitive, synergistic, or combinatorial) stimulation with agrin, neuregulin, and ACh.